WO2010030203A1 - Anticorps monoclonal dirigé contre la transthyrétine humaine amyloïdogène et des formes modifiées de celle-ci et son utilisation dans la détection et le traitement de la fap et de pathologies présentant une ttr modifiée - Google Patents

Anticorps monoclonal dirigé contre la transthyrétine humaine amyloïdogène et des formes modifiées de celle-ci et son utilisation dans la détection et le traitement de la fap et de pathologies présentant une ttr modifiée Download PDF

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WO2010030203A1
WO2010030203A1 PCT/PT2008/000034 PT2008000034W WO2010030203A1 WO 2010030203 A1 WO2010030203 A1 WO 2010030203A1 PT 2008000034 W PT2008000034 W PT 2008000034W WO 2010030203 A1 WO2010030203 A1 WO 2010030203A1
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Prior art keywords
ttr
monoclonal antibody
mab
fap
modified
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PCT/PT2008/000034
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English (en)
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Maria João SARAIVA
Anabela Clao Teixeira
António Bernardino GUIMARÃES PARADA
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Biocodex - Incubação De Empresas De Ciências Da Vida, S.A.
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Priority to PCT/PT2008/000034 priority Critical patent/WO2010030203A1/fr
Publication of WO2010030203A1 publication Critical patent/WO2010030203A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease

Definitions

  • the present invention relates to a monoclonal antibody (mab) for human mutant transthyretin (TTR) methods for its production and its uses such as in screening for familial amyloidotic polyneuropathy (FAP) , pathologies presenting modified forms of TTR and in research and treatment of associated diseases thereof. Therefore, the present invention applies to pharmaceutical and medical areas.
  • mab monoclonal antibody
  • TTR transthyretin
  • FAP familial amyloidotic polyneuropathy
  • TTR transthyretin
  • TTR is the main constituent of extracellular amyloid deposits in FAP, found in several organs and tissues, in particular, in nerve. TTR is produced mainly by the liver and choroid plexus, and can be found in the plasma and cerebrospinal fluid, where it circulates as a tetramer composed of four 127 amino acid sub-units. TTR has a physiological role in the transport of the hormone thyroxine (T 4 ) and of retinol (indirectly, through RBP - Retinol Binding Protein) .
  • T 4 the hormone thyroxine
  • retinol indirectly, through RBP - Retinol Binding Protein
  • T 4 binds in a central channel formed by the interaction of the four monomers (Blake et al., 1976) and RBP attaches to the surface of the tetramer, as revealed by the structure of the complex (Monaco et al . , 2002) .
  • Each TTR monomer is composed of eight ⁇ -strands designated A-H, organised in 2 ⁇ -sheets interacting in a face-to- face fashion, forming the hydrophobic core of the molecule.
  • Sheets CBEF are oriented to the exterior of the molecule and sheets DAGH line the T 4 binding channel, with exclusively hydrophobic residues.
  • TTR variants along the peptide chain identified a peak in the distribution of TTR variants along the peptide chain corresponding to a region of maximum mutational frequency ("hot-spot"), associated with the edge strands C and D (residues 45-58) of the two ⁇ -sheets that form the structural framework of the TTR molecule. Changes at the edges of the ⁇ -sheets of TTR could be so significant as to form, or expose, an amyloidogenic determinant, not present in the native TTR protein.
  • hot-spot maximum mutational frequency
  • Leu55Pro TTR has been associated with early onset and highly aggressive amyloidosis and X-ray structural data showed a crystal with intermolecular contacts profoundly altered, resulting in the assembly of an oligomeric structure that might represent an intermediate in the amyloidogenesis cascade (Sebastiao et al., 1998) .
  • the exchange of the amino acid Leu for the amino acid Pro at position 55 changes the TTR secondary structure by disruption of strand D; the molecule is rearranged and residues 54-56 are included in a long loop that connects ⁇ strands C and E, which is involved in the crystallographic packing observed in this crystal structure.
  • This "amyloid-like" structure revealed additional positional differences such as the contacts of the ⁇ -helix and the AB loop.
  • structural comparative studies of TTR native fold and the abnormal conformation of Leu55Pro variant revealed that the OH group of Tyr 78 plays an important role in maintaining the tertiary structure of the AB loop, which to the design the TTR mutant Tyr78Phe (Redondo et al . , 2000) . It was found that this variant formed fibrils in the same pH range as Leu55Pro TTR and was reactive, in its tetrameric soluble form, to a monoclonal antibody described by Goldsteins and co-workers (1999) as specific for amyloid.
  • a substitution of a Tyr for a Phe at position 78 might have caused a rearrangement in the tight tetrameric structure due to the breaking of key H-bonds, yielding a soluble amyloidogenic intermediate with exposed cryptic epitopes, present in an earlier step of the amyloidogenic process .
  • mutant TTR can deposit early in an aggregated non-fibrilar form in asymptomatic carriers of TTR Val30Met, or in transgenic mice carrying this mutant human gene (Sousa et al . , 2002), before overt amyloid deposition.
  • Deposition of the protein in this oligomeric, non- fibrillar form triggers the expression of inflammatory molecules (such as interleukine 1) and of oxidative stress molecules (such as nitric oxide synthase) (Sousa et al . , 2001a) .
  • inflammatory molecules such as interleukine 1
  • oxidative stress molecules such as nitric oxide synthase
  • Monoclonal antibodies (mabs) produced in mice against highly- aggressive amyloidogenic synthetic TTR mutants were shown to react with high molecular weight TTR aggregates, and do not recognize soluble native TTR when tested under ELISA (enzyme-linked immunoassay) by direct procedures. It was hypothesized that these mabs recognize cryptic epitopes that are exposed in mutant TTRs resembling aggregated TTR. Interestingly, these mabs react to TTR from plasma of FAP patients and/or asymptomatic carriers of neuropathic TTR mutants, but not to plasma from normal individuals (Palha et al., 2001) .
  • the present invention discloses discriminating properties between mutant and normal TTR not only in direct, but also in indirect ELISA procedures. Furthermore, the mab TTR of the present invention detects by immunoblotting particular modified TTR bands in plasma of FAP patients. The presence > of modified forms of TTR in plasma has been speculated in the literature, but never documented to date, opening, for the first time the possibility to address pathogenesis and follow up of therapies in FAP.
  • TTR has been associated with a number of pathological conditions, including different types of cancers (Fung et al 2005, Moore et al . 2006, Escher et al . , 2007) , Alzheimer and other neurodegenerative diseases (Biroccio et al , 2006) , pre-eclampsia (Vascotto et al , 2007), moyamoya disease (R ⁇ ggeberg et al . , 2008); in these conditions, modified forms of TTR are detected in plasma, cerebrospinal fluid or amniotic fluid (for pre-eclampsia) , most of them representing oxidized TTR.
  • the used techniques require cumbersome procedures, expensive sophisticated mass spectrometry equipment and expertise not available in many institutions and laboratories, limiting their use on a routine basis .
  • the present invention allows the detection of these modifications, such as oxidation, in a one dimensional electrophoretic separation followed by Western bloting with the mab, opening the possibility of the use of the mab as a biomarker in these pathologies.
  • the present invention features a mab produced in mice against recombinant mutant Tyr78Phe TTR mutant that, selectively recognises under specific conditions: (i) plasma samples from carriers of certain known TTR mutants associated with FAP; (ii) slow migrating electrophoretic TTR band only present in the plasma of carriers of mutant TTR; (iii) faster migrating electrophoretic TTR bands detected in pathologies presenting modified TTR; the mab is able to remove TTR modified species from tissues of transgenic mice for human mutant Val30Met TTR presenting TTR extracellular deposition.
  • a second aspect of the present invention refers to a method to generate such preferred mab.
  • the method involves (a)hybridoma production by fusion of spleens cells sample from immunized mice with Sp2/0 cells sample; (b) screening of hybridomas reactive to different mutant TTRs and non-reactive to normal non-mutated TTR, by ELISA immunoassay under native conditions; (c) re-cloning of the mab; and (d) isolation and sequence determination.
  • a third aspect of the present invention refers to a kit for the detection of FAP comprising the monoclonal antibody, according to the present invention.
  • a fourth aspect of the present invention refers to the use of the produced mab, according to the present invention, to detect the presence of known mutant amyloidogenic TTR in plasma samples and, for unknown mutant TTRs.
  • the mab of the invention is useful as to give indication for the presence in plasma of an amyloidogenic mutation.
  • the mab also finds use in screenings for epidemiological studies of TTR amyloidogenic mutations in the population once this mab has unique properties as recognizing cryptic epitopes/conformations associated with circulating plasma mutant TTR species, not present in normal TTR, which, to date, have never been documented.
  • a fifth aspect of the present invention is the use of the mab for detection of faster migrating electrophoretic TTR bands in pathologies presenting modified TTR, making the mab useful in biomarker studies associated with these pathologies.
  • mice Compared to age matched control non- immunized mice, Tyr78Phe immunized mice had a significant reduction in TTR deposition usually found in this strain, in particular in stomach and intestine; by contrast, animals immunized with Val30Met did not show differences in deposition in comparison with non- immunized mice.
  • the monoclonal antibody to human amyloidogenic transthyretin comprises the following amino acid residues: SEQ. ID 1: Amino acid sequence of AD7F6 antibody VL regions)
  • SEQ. ID 2 Amino acid sequence of AD7F6 antibody VH regions
  • SEQ. ID 3 Nucleotide sequence of full-length heavy chain
  • SEQ. ID 4 Nucleotide sequence of full-length light chain
  • the monoclonal antibody of the invention produces an IgG monoclonal antibody of subclass IgGa b* kappa light chain.
  • the antibody has a molecular weight of 160 kDa, which upon reduction yields 50 kDa and 28 kDa fragments. It recognizes amyloidogenic plasma mutant TTR both in reducing and non- reducing conditions and modified plasma TTR. These properties are assigned to unique sequences of the mab that reacts with a cryptic epitope in TTR that is exposed by mutant and/or modified TTR.
  • the present invention features a method to generate such preferred mab. This method involves the following steps:
  • the mab reacts in ELISA immunoassay under native conditions with Val30Met TTR in its isolated form obtained either from the periplasmic space of recombinant bacteria or from the plasma of carriers of Val30Met but does not react with normal, non-mutated TTR isolated from bacteria or plasma.
  • the monoclonal antibody of the present invention reacts with modified human plasma TTR from carriers of mutant TTR Val30Met.
  • the mab of the invention is useful as to give indication for the presence in plasma samples of an amyloidogenic mutation.
  • the mab of the present invention is applicable in the detection of known mutant amyloidogenic TTR in plasma samples and, for unknown mutant TTRs, to give indication for the presence in plasma of an amyloidogenic mutation.
  • the mab also finds use in screenings for epidemiological studies of TTR.
  • the mab of the invention detects abnormal TTR bands representing modified non-mutated TTR, making the mab useful in biomarker studies associated with pathologies presenting modified TTR.
  • One particular embodiment, related to the use of the mab according to the present invention, is the detection of amyloidogenic TTR mutants in plasma and involves the production of an ELISA kit comprising the mab of the present invention and the use of the following method:
  • the mab reacts in ELISA immunoassay under native conditions with Val30Met TTR in its isolated form obtained either from the periplasmic space of recombinant bacteria or from the plasma of carriers of Val30Met but does not react with normal, non-mutated TTR isolated from bacteria or plasma.
  • the mutant TTR is isolated from the periplasmic space of recombinant bacteria producing:
  • the mab of the invention detects synthetically produced oligomeric or aggregated forms of TTR.
  • One particular method of detection comprises :
  • the mab of the invention detects a slower migrating electrophoretic band -SMT- present in plasma samples of carriers of Val30Met TTR, composed of intact modified TTR, absent in normal plasma samples.
  • a slower migrating electrophoretic band -SMT- present in plasma samples of carriers of Val30Met TTR, composed of intact modified TTR, absent in normal plasma samples is detects.
  • SMT is detected by:
  • the mab of the invention detects two faster migrating electrophoretic bands FMT - generated upon oxidation treatment of plasma samples of carriers, of Val30Met TTR, or normal controls.
  • One particular method of detection involves:
  • FMT are detected in isolated TTR from plasma samples or recombinant bacteria after oxidation.
  • FMT are detected in plasma of pathologies presenting modified TTR.
  • the mab can remove TTR deposits from transgenic mice for human Val30Met TTR (hTTR Met30) .
  • One particular method comprises:
  • Val30Met TTR as means to elucidate mechanisms of deposition and action of drugs on the mutant human
  • Figure 1 Represents the comparison of the mab reactivity with the TTR isolated from plasma of Val30Met carriers and from control non-carriers individuals and, the reactivity to Val30Met -TTR obtained from recombinant bacteria.
  • Figure 2 Represents the specificity of the mab towards oligomeric and aggregated forms of TTR.
  • Figures 3a and 3b Represent the comparison of the mab reactivity towards plasma of Val30Met carriers and of control non-carriers individuals by direct (3a) and sandwich (3b) ELISA procedures.
  • Figures 4a and 4b Represent the detection of soluble modified slow migrating TTR (arrow) in human sera of Val30Met carriers (4b) and in sera from transgenic mice (4a) for the same mutant after native Western blotting using the mab.
  • Figures 5 Represent the demonstration of the slow migrating TTR (SMT) in sera of carriers of Val30Met TTR (5a) and of fast migrating TTR (5b) (FMT) after oxidation treatment following immunoprecipitation and Western blotting from a denaturant gel, using the mab.
  • SMT slow migrating TTR
  • Figure 6 Represents the demonstration of the effect of the mab by passive immunization on TTR deposition in the sciatic nerve of transgenic mice.
  • TTR was expressed in E. coli strain Bl-21 after transformation with individual expression plasmids containing WT or mutated TTR (Val30Met and Tyr78Phe) and the periplasmic contents were obtained by osmotic shock. The supernatant was fractionated on DEAE-cellulose, as described by Almeida and colleagues and TTR containing peaks were dialysed against water and lyophilised. Further purification was achieved by preparative gel electrophoresis, in a native Prosieve agarose system (FMC, Mockland, ME) . TTR was purified from serum following an established procedure (Almeida et al .
  • Val30Met TTR and Tyr78Phe TTR (1 mg ml "1 ) were incubated in 0.05 M sodium acetate/0,1 M KCl buffer pH 3.7, for 48 hours, at room temperature, to form amyloid fibrils. Oligomers were produced by stirring at room temperature (Teixeira et al., 2006) The preparations were positive by Thioflavin-T spectrofluorometric assays.
  • mice used in the immunization were 16 months old TTR knockouts obtained from the Jackson's laboratory and were crossed to the 129Sl/Sv background for more than 10 generations, which were immunised with the recombinant mutant TTR Tyr78Phe by injecting 10 ⁇ g of antigen diluted in Freund's complete adjuvant (Sigma) intraperitonealy, in a final volume of 200 ⁇ l . Two weeks later, a new immunisation was performed, injecting 10 ⁇ g of antigen diluted in Freund's incomplete adjuvant (Sigma), in a final volume of 200 ⁇ l . This procedure was repeated twice every two weeks, until high levels of IgG reactivity was detected in ELISA assays.
  • Spleen was homogenised in RPMI -1640 medium (Gibco BRL) , spun and then re-suspended in cold NH 4 Cl (0.17 M) . After a short spin the pellet was mixed with approximately 10 7 SP2/0 myeloma cells, spun, re-suspended in a small volume and incubated at 37 0 C. To induce cell fusion, a 50% PEG solution (Sigma) was added drop-wise for one minute and the mixture re-suspended in RPMI-1640 medium. After a final spin, the supernatant was discarded and cells were re-suspended in foetal bovine serum (FBS, Gibco BRL) .
  • FBS foetal bovine serum
  • This suspension was then diluted in complete selection medium [RPMI -1640 supplemented with HEPES, L-Glutamine, FBS (10%) and hypoxanthine/aminopterin/thymidine (HAT) (GibcoBRL) ] to a final volume of 100 ml and seeded in microtiter plates (96 wells, Nunc, Denmark), 200 ⁇ l/well.
  • the plates were left to incubate for five > days at 37 0 C in a 5% CO 2 humidified incubator. Feeding was then performed every third day, for one week, with the same medium. On day 7, the feeding protocol was repeated with hypoxanthine/thymidine (HT) selection medium and plates were returned to the incubator and checked every two or three days.
  • complete selection medium [RPMI -1640 supplemented with HEPES, L-Glutamine, FBS (10%) and hypoxanthine/aminopterin/thymidine (HAT) (GibcoBRL) ] to a final volume
  • microtiter plates (96 wells, Nunc, Denmark) were coated with 1 ⁇ g of TTR or 100 ⁇ l of serum diluted 1:10 in coating buffer (0.1 M carbonate buffer, pH 9.6), using 100 ⁇ l/well. Washes were performed three times with PBST (phosphate buffered saline, 0.02% Tween) and twice with PBS. After blocking with 5% non-fat dry milk in PBS, 200 ⁇ l/well and washed as mentioned above, plates were incubated with 100 ⁇ l/well of undiluted hybridoma culture supernatants, for one hour at room temperature.
  • coating buffer 0.1 M carbonate buffer, pH 9.6
  • Protein G Sepharose High performance (MabTrap G II protein G Pharmacia) was used for purification and isolation of monoclonal antibody from cell culture supernatants .
  • Mab was eluted from affinity column with Immuno Pure Gentle Elution Buffer (Pierce) .
  • Proteins were analysed either in native or denaturing conditions. Native electrophoresis was carried out on 10% (w/v) acrylamide gels system. Electrophoresis under denaturing conditions was performed in SDS-PAGE gels (15% acrylamide, 0.1% (w/v) SDS), after heat-treatment of samples and addition of ⁇ -mercaptoethanol (0.1 M) . Proteins were transferred from gels into nitro-cellulose membranes (HybondTM-C pure, Amersham) , using a Tris -Glycine system, for one hour, at 1 mA/ctn 2 of membrane.
  • Native electrophoresis was carried out on 10% (w/v) acrylamide gels system. Electrophoresis under denaturing conditions was performed in SDS-PAGE gels (15% acrylamide, 0.1% (w/v) SDS), after heat-treatment of samples and addition of ⁇ -mercaptoethanol (0.1 M) . Proteins were transferred from gels into nitro-cellulose membranes (Hybond
  • the immunodetection was performed with either a rabbit anti-TTR polyclonal antibody (DAKO, 1:1000 dilution), for one hour at room temperature and goat anti- rabbit immunoglobulins, HRP conjugated (Pierce, 1:5000 dilution), or the mab (pure hybridoma supernatants) , for one hour at room temperature and sheep anti-mouse immunoglobulins-HRP (Pierce, 1:5000 dilution) .
  • TTR was visualised using either the ECL method (Pierce) or DAB substrate.
  • Membranes were saturated in 5% skim milk in PBS 1 hour at RT followed by incubation 1 hour with monoclonal antibody (pure hybridoma supernatant) and sheep anti -mouse immunoglobulin G - HRP conjugated. Proteins were visualised with DAB.
  • TTR from sera of Val30Met carriers was semi-purified by- affinity chromatography to isolate the slow TTR migrating (SMT) band detected by the mab on western blotting.
  • SMT slow TTR migrating
  • DAKO rabbit anti-TTR polyclonal antibody
  • N- terminal sequencing of the SMT band was performed after SDS transfer to PVDF membrane and Coomassie staining.
  • the fast migrating band (FMT) detected by the mab on western blotting after oxidation was characterized by mass spectrometry of tryptic digests and N- terminal sequence, as described above, after application of 5 ⁇ g of oxidized TTR to a SDS gel which was silver stained, followed by excision of the FMT band.
  • This protocol utilizes a signal sequence primers based on results from N-terminal sequence and specific gamma 2B or kappa reverse primers for PCR amplification of mouse immunoglobulin genes using l sC strand cDNA, generated from hybridoma total RNA, as template.
  • RNA was prepared using the Tryzol reagent (Invitrogen) according to manufacturer's specifications. Briefly, 2 ml of Tryzol reagent was added to semi -confluent T-175 flask containing hybridoma cells and pipeted repeatedly to lyse the cells. The lysate was transferred to two centrifuge tubes and 0.2 ml of chlorophorm was added to each. After vigorous mixing the tubes were spun to separate the mixture into phases . The upper aqueous phase containing the RNA was transferred to fresh, tubes and precipitated with 1 ml of isopropanol. After centrifugation, the pellets were washed with 75% ethanol, and resuspended in 100 ⁇ l of DEPC-treated water.
  • Tryzol reagent Invitrogen
  • RNA sample was generated using MMLV reverse transcriptase according to manufacturer' s specifications (Clontech, Advantage RT for PCR) .
  • Two microliters of RNA were used as template in a reaction containing oligo-dT primer.
  • the template and primer Prior to assembly of the reaction, the template and primer were briefly incubated at 70 0 C for denaturing of template and rapid annealing of the primer.
  • To this mix 4 ⁇ l of 5x reaction buffer, 1 ⁇ l of 10 mM dNTPs. 0.5 ⁇ l of RNAse inhibitor, and 1 ⁇ l of RT was then added.
  • the reaction was run for 1 hour at 42 0 C.
  • the RT was then inactivated by heating at 94 °C for 5 minutes, then placed on ice.
  • the cDNA preparation was then stored at -20 0 C for use in PCR.
  • PCR reactions were assembled using the Expand PCR kit (Boerhinger Mannheim) according manufacture's specifications. Successful heavy chain amplification generated products of approximately 1.4 Kb in size. Successful light chain amplification generated products of approximately 0.7 Kb in size .
  • the pGEM-T easy vector System (Promega) was used for the cloning of PCR products obtained above according manufacture's specifications.
  • the PCR product to be ligated was gel purified using QIAEX kit (QIAGEN) .
  • QIAEX kit QIAGEN
  • Transformation of ligated pGEM-T vector and PCR products were performed using XL2-Blue Ultracompetent cells (Statagene)
  • EXAMPLE 1 The mab does not recognize soluble normal TTR but rather amyloidogenic TTR in its soluble and/or aggregated form.
  • TTR aggregates e.g., prepared from Tyr78Phe or Val30Met
  • Val30Met carriers include asymptomatic and symptomatic individuals.
  • EXAMPLE 2 The mab detects modified soluble TTR species with altered electrophoretic mobility in plasma from carriers of the Val30Met TTR mutation.
  • Figure 5B shows sera from Val30Met carriers and controls pre- treated under oxidative conditions before immunoprecipitation by the mab followed SDS-PAGE and Western blotting using the same mab; a faster migrating TTR (FMT) band is disclosed both in mutant carriers and normal controls.
  • FMT is intact TTR, as assessed by N-terminal sequence and MALDI-TOF mass spectrometry after tryptic digestion and represents TTR with modifications that alter electrophoretic mobility.
  • EXAMPLE 3 Passive immunization with the mab of transgenic mice for human Val30Met TTR, presenting tissue TTR deposition, results in removal of deposition.
  • the mab was administrated for 6 months to young 3 months old transgenic mice for human Val30Met TTR in a background deficient for heat-shock factor 1; this strain of mice normally presents deposition of human TTR in the peripheral nervous system, particularly in the nerve starting at 3 months of age,- at 9 months deposition in the nerve is highly penetrant.
  • the animals receiving passive immunization through mab treatment presented virtually no TTR deposits as compared to mice receiving vehicle alone, as documented by semiquantitative immunohistochemistry in Figure 6.

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Abstract

La présente invention concerne un anticorps monoclonal (AcM) dirigé contre une transthyrétine (TTR) mutante humaine, un kit de détection de la PAF comprenant l'AcM susmentionné, des procédés pour sa production ainsi que ses utilisations, notamment dans le dépistage de la polyneuropathie amyloïde familiale (PAF) et de pathologies présentant des formes modifiées de TTR, et dans la recherche et le traitement de maladies associées à celle-ci. Cet anticorps monoclonal présente comme caractéristique principale la reconnaissance de TTR mutante plasmatique amyloïdogène et de TTR modifiée. Ces propriétés sont attribuées à des séquences uniques de l'AcM réagissant avec un épitope cryptique dans la TTR qui est exposé par TTR mutante et/ou modifiée, d'où une spécificité élevée pour les formes modifiées de TTR plasmatique mutée et non mutée. L'AcM de l’invention trouve une utilité dans des études épidémiologiques de mutations amyloïdogènes de TTR dans la population et dans des études de biomarqueur associées à des pathologies présentant une TTR modifiée.
PCT/PT2008/000034 2008-09-09 2008-09-09 Anticorps monoclonal dirigé contre la transthyrétine humaine amyloïdogène et des formes modifiées de celle-ci et son utilisation dans la détection et le traitement de la fap et de pathologies présentant une ttr modifiée WO2010030203A1 (fr)

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Cited By (15)

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WO2014124334A3 (fr) * 2013-02-08 2014-12-24 Misfolding Diagnostics, Inc. Anticorps anti-transthyrétine et leurs utilisations
WO2015092077A1 (fr) * 2013-12-20 2015-06-25 Neurimmune Holding Ag Thérapie de l'amyloïdose de la transthyrétine (ttr) à base d'anticorps, et anticorps d'origine humaine afférents
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